A class I PI3K signalling network regulates primary cilia disassembly in normal physiology and disease

Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA-related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kβ produce the PIP3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered ‘Disorders with Ciliary Contributions’, a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction.

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Antibodies
Antibodies used from the corresponding author upon request.
Human participants were not used in this study.
As per box 1 above, race, ethnicity or other social groupings are not relevant to this study which did not use human participants.
As per box 1 above, population characteristics are not relevant to this study which did not use human participants.
As per box 1 above, recruitment is not relevant to this study which did not use human participants.
As per box 1 above human ethics oversight is not relevant to this study which did not use human participants.
Samples sizes were chosen based on our and our collaborators prior experience and the standards used in the field.
No data were excluded form the analysis.
All experiments were performed for at least n=3 biological replicates as indicated in the figure legends.
Mice were allocated to groups based on their genotype.Randomization is not relevant to the cell biology experiment presented here.
Data from mouse embryos was collected prior to genotyping, therefore investigators were blinded.The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of of group (a (a 'group' is is an an analysis of of identical markers).
All plots are contour plots with outliers or or pseudocolor plots.
A numerical value for number of of cells or or percentage (with statistics) is is provided.

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Cell population abundance
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As As per box 1 above, novel plant genotypes are not relevant to to this study which did not use plants.
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As As per box 1 above, plant authentication is is not relevant to to this study which did not use plants.
For cell cycle analysis of of BPH1, cells were collected in in PBS, fixed in in 4% 4% PFA for 15 15 min at at room temperature and permeabilized with 0.2% Triton X-100 in in PBS for 30 30 min at at room temperature.Then, pelleted cells were stained with propidium iodide solution (2% PI PI and 0.1 mg/ml RNAse A in in PBS) for 30 30 min at at 37ºC and analysed by by FACS (BD Biosciences).
For cell cycle analysis of of MEFs, MEFs were treated as as described in in the Methods.Staining of of samples was performed with a BrdU Flow Kit according to to the manufacturer (BD Pharmigen) instructions.
For cell cycle analysis of of BPH1 a BD BD Biosciences Instrument was used.For cell cycle analysis of of MEFs an an CyAn ADP (DakoCytomation) was used.
For cell cycle analysis of of BPH1 FlowJo v10.10 was used.For cell cycle analysis of of MEFs Summit V4.0 (DakoCytomation) was used.
Cell population abundance does not apply to to these experiments.
For cell cycle analysis of of BPH1.Forward versus side scatter density plots (FSC vs vs SSC) gating was used identify cell population of of interest based on on size and granularity and to to exclude debris .A forward scatter height (FSC-H) vs. forward scatter area (FSC-A) density plot was then used to to exclude doublets.A single parameter histogram was next used to to further identify cell population.
For cell cycle analysis for MEFs.An An initial gate was set in in the FS/SS plot to to exclude cell debris.A second gate was set to to select cells with 2n 2n and 4n 4n DNA to to exclude doublets and clumps.Cells with fluorescence intensity higher than 10^1 were considered BrdU positive.